The nucleolus is a remarkable nuclear organelle that is conserved from yeast to man and plays numerous roles in the life of normal and tumor cells. The nucleolus harbors the genes and machinery required to synthesize and to assemble ribosomes. Misregulation of this process through the absence of RB, for example, is a hallmark of cancer. In addition, recent findings suggest that proliferation factors (e.g. MDM2) and cell-cycle activators (e.g. Cdc14) are sequestered in the nucleolus until appropriate signals stimulate their release. Tumor cells are often defective in this nucleolar sequestration step, underscoring the role of the nucleolus in cancer. Despite the importance of the nucleolus, the factors responsible for its unique structure are poorly understood. In the yeast, Saccharomyces cerevisiea, the nucleolar factor, Neti may be a key player in the structure and function of the nucleolus. Neti regulates the cell cycle, nucleolar chromatin structure, and nucleolar architecture. The goals of this project are to characterize the nature of the interaction of Net1 with the nucleolus and to determine the role of Net1 in nucleolar structure. Such an understanding of the activity of Neti may lead to significant insights into the structure of the nucleolus and the role of the nucleolus in the cell cycle and cancer. The aims of this project are to ascertain the nucleolar DNA sequences that interact with NetI using chromatin immunoprecipitation assays. In addition, DNA binding studies will be used to determine if Net1 binds directly to nucleolar DNA. If Net1 binding is indirect, then mutant screens and immunofluorescence will be used to identify binding partners. Finally, specialized nucleolar DNA plasmids will be utilized with immunofluorescence and chromatin immunoprecipitation assays to explore the steps in the assembly of the nucleolus that require Net1.